Halt phosphatase inhibitor cocktail 100x

Cells were washed in PBS and incubated for 15 minutes at 4°C and in NADOC (0.5% Tritonx-100, 0.5% sodium deoxycholate, 50 mM Tris, and 150 mM NaCl) lysis buffer (Thermo Fisher Scientific) with protease (HaltTM Protease Inhibitor Cocktail 100X, Thermo Fisher Scientific) and phosphatase inhibitors (HaltTM Phosphatase Inhibitor Cocktail 100X, Thermo Fisher Scientific). The extracts were centrifuged at 14,000g for 15 minutes. Protein concentrations in the supernatant were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific) and a Tecan Safire® plate reader. Protein extracts (25 μg) were resolved by 4-12% SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes (iBlot, Invitrogen). Membranes were blocked with SEABLOCK blocking buffer (Thermo-Scientific) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies diluted in blocking buffer. After washing in PBS-Tween buffer, the membranes were incubated with secondary antibodies conjugated to IRDye fluorophores. The infrared signal of the membranes was detected using an Odyssey detection system (Li-Cor biosciences).

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was obtained from Avanti Polar Lipids (Alabaster, Alabama, United States). Cholesterol (Cholesterin®) was obtained from Riedel-de-Haën™ (Honeywell, Germany). miR-1296 mimic (accession no. MIMAT0005794) and control miRNA (NC) were purchased from Life Technologies (Carlsbad, California, United States). Fluorescently labeled miR-1296-CY3 conjugated was purchased from Invitrogen (Carlsbad, California, United States). Quant-iT™ RiboGreen® RNA Assay Kit was purchased from Invitrogen (Carlsbad, California, United States). HaltTM Phosphatase Inhibitor Cocktail 100X and HaltTM Protease Inhibitor Cocktail 100X were purchased from Thermo Scientific (Waltham, Massachusetts, United States). All primary antibodies, including CCND1 (cat no. 2978S), PARP1 (cat no. 2532S), and housekeeping gene antibody GADPH (cat no. 5174S), were purchased from Cell Signalling (Massachusetts, United States). Horseradish Peroxidase (HRP) conjugated mouse IgG kappa binding protein (m-IgGκ BP, cat no. sc-516102) was obtained from Santa Cruz Biotechnology (Texas, United States).